The electrophoretic display (EPD) has attracted widespread attention due to its great visual perception, energy-saving, portability, and bistability. However, the EPD still has many problems in response time, colorization, etc., which limits its practical application. In this paper, novel blue electrophoretic particles were prepared with copper (II) phthalocyanine and high ionization 1-butyl-1-methyl piperidinium bromide mono ionic liquid. It was shown that electrophoretic particles dispersed in a non-polar tetrachloroethylene medium had high Zeta potential and electrophoretic mobility. At the same time, electrophoretic particles showed better dispersion stability. Finally, the prepared blue electrophoretic particles and white titanium dioxide particles were compounded to prepare blue and white dual-color electrophoretic dispersion. An EPD cell was made to test its performance. The results showed that the prepared blue and white dual-color electrophoretic dispersion could realize a reversible response. Piperidine mono ionic liquid increased the surface potential of copper (II) phthalocyanine from +30.50 mV to +60.27 mV, enhancing it by 97.61%. Therefore, we believed that modifying particles with high ionization mono ionic liquid had great applicability to the modification of electrophoretic particles, and blue particles prepared with piperidine mono ionic liquid as a charge control agent (CCA) were excellent candidates for EPDs. Because of its stable image, wide viewing angles, good contrast ratio, and low power consumption, it is generally regarded as a green electronic display ,2. Compaifficult . Copper ifficult ,15,16,17The key to the excellent dispersibility and electrophoretic performance of electrophoretic particles lies in their surface charge in low dielectric constant non-polar medium. Therefore, the modification of electrophoretic particles also focuses on improving their dispersion stability and surface charges. In recent years, there have been reports of treating pigment particles with anionic surfactants, cationic surfactants, non-ionic surfactants, or hyperdispersants to improve their dispersibility and surface charges ,23,24,25In this article, a method of preparing blue electrophoretic particles was proposed. We used high ionization 1-butyl-1-methylpiperidinium bromide mono ionic liquid as a charge control agent to modify copper (II) phthalocyanine (CuPc) pigment particles. Finally, blue and white dual-color E-ink was prepared by mixing and dispersing blue electrophoretic particles and white negatively charged titanium dioxide particles in tetrachloroethylene.Copper (II) phthalocyanine (CuPc) (99%), 1-butyl-1-methyl piperidinium bromide (99.0%), potassium bromide, and tetrachloroethylene (TCE)  were all purchased from Macklin . Anhydrous ethanol was purchased from Tianjin Damao . Sorbitan monooleate (Span 80) and titanium dioxide (98%) were purchased from Aladdin . All reagents could be used without further purification. Ultrapure water was used in the whole experiment. Two pieces of 3 cm × 6 cm indium-tin oxide (ITO) transparent conductive glasses (90 ± 20 Ω/sq) were purchased from Shenzhen Laibao High Tech Co., Ltd., . Torr Seal was purchased from Agilent Technologies .A total of 50 mL of anhydrous ethanol and 0.1 g of 1-butyl-1-methyl piperidinium bromide (IL) were added to a 100 mL flask containing 0.5 g CuPc and vibrated with ultrasound for 15 min. The mixture was refluxed by stirring at 50 °C for 1.5 h, and then the reactant was removed at 70 °C with a vacuum dryer. CuPc modified with 1-butyl-1-methyl piperidinium bromide (CuPc-IL) was obtained.A total of 2 mg CuPc-IL and 0.7 mg Span 80 were added to 10 mL TCE in a flask. Then, the blue electrophoretic dispersion was obtained by ultrasonic dispersion for 40 min. This resulted in a blue electrophoretic dispersion.A total of 2 mg CuPc-IL, 1 mg titanium dioxide, and 0.1 mg Span 80 were added to 10 mL TCE in a flask. The blue and white dual-color electrophoretic dispersion was obtained by ultrasonic dispersion for 40 min.The conductive surfaces of the two ITO glasses were bonded together with sealant, leaving a 1 mm high gap in the middle. The blue and white dual-color EPD cell was obtained by injecting the blue and white electrophoretic dispersion into the cell with a syringe.−1 at 25 °C.Energy dispersive spectroscopy (EDS)  was used to characterize the elemental composition of CuPc and CuPc-IL. CuPc, CuPc-IL, IL, and potassium bromide powder were dried for 5 h in a 70 °C vacuum oven to prepare potassium bromide tablets, respectively. The functional groups and chemical bonds of the samples were characterized by Fourier transform infrared spectroscopy (FT-IR)  in the wavelength range of 400–4000 cmAn appropriate amount of CuPc-IL and CuPc was dispersed into tetrachloroethylene and treated with ultrasound for 30 min. Zeta potential and particle size were then measured by Zeta potential and a particle size analyzer  at 25 °C. CuPc, CuPc-IL, and IL were dried in an oven at 70 °C for 3 h. Under a nitrogen atmosphere, samples were produced at a heating rate of 5 °C/min from 30 °C to 800 °C by using a thermal analyzer .An experimental platform was developed to test the blue and white dual-color EPD cell. It was composed of a driving system and a testing system. The driving system was composed of a function generator  and a voltage amplifier , used to generate driving waveforms. The testing system was composed of a computer  and a colorimeter , which was used to record the CIE Yxy chromaticity diagram of the EPD cell.The modification process of copper (II) phthalocyanine could be simplified . The priElement composition was detected by an energy spectrometer . The two−1, which was the aromatic C-H of the phthalocyanine ring. Peaks at 1612.5, 1498.7, 1421.5, 1334.7, 1286.5, and 1091.7 cm−1 were stretching vibration bands of plane C-C or C-N of phthalocyanine ring. Peaks at 1166.9 and 1120.6 cm−1 were in-plane bending vibration bands of a C-H of the benzene ring. Peaks at 871.8, 754.1, and 723.3 cm−1 were out-of-plane bending vibration bands of a C-H of the benzene ring, and 900.7 cm−1 was a stretching vibration peak of Cu-N. At the same time, out-of-plane bending vibration bands of benzene rings also appeared in the range of 400–700 cm−1. The FT-IR spectrum of IL displayed the characteristic peaks at 2952.9 and 2869.9 cm−1, which were stretching vibration peaks of C-H in alkyls or piperidinium. At 1460 cm−1 was the plane bending vibration peak of methyl and methylene. Noted at 945.1 and 904.5 cm−1 were the out-of-plane bending vibration peaks of C-H in alkyls or piperidinium; Br- had no absorption peak. In the infrared spectrum of CuPc-IL, there were characteristic absorption peaks of both CuPc and IL, and the aliphatic C-H peak of IL was decreased by combining with CuPc. Therefore, the results indicated that IL was coated on the surface of the CuPc particles.The chemical functional groups of CuPc, CuPc-IL, and IL were characterized by Fourier Transform infrared spectroscopy (FT-IR) . The FT-During the Thermogravimetry analysis (TGA) curve , CuPc, CThe Zeta potential of particles is caused by the adsorption of relevant ions on the pigment surface since the Zeta potential of modified particles affects the electrophoretic speed of particles in the electrophoretic medium. Therefore, the modification of electrophoretic particles is mainly to improve the Zeta potential and thus improve electrophoretic mobility. The test results showed that 1-butyl-1-methyl piperidinium bromide had an obvious effect on improving the Zeta potential of CuPc; the surface potential was nearly doubled from +30.50 mV to +60.27 mV . CompareAfterward, 1-butyl-1-methylpiperidinium bromide single ionic liquid was used and compared with 1-butyl-3-methylimidazole bromide single ionic liquid used by Eshkalak  et al. a+ in the cations of pyrrolidine and piperidine ionic liquid was a stable structure of ammonium salt, which led to easier ionization than imidazole ionic liquid. Moreover, the modification effect of piperidine ionic liquid was better than that of pyrrolidine ionic liquid because the six-membered ring was more stable than the five-membered ring, and the ionization effect of piperidine ionic liquid was better than that of pyrrolidine ionic liquid. Therefore, the potential of CuPc particles modified with piperidine ionic liquid could be further increased [The reason why the modification effect of pyrrolidine ionic liquid and piperidine ionic liquid was better than that of imidazole ionic liquid was that Nncreased ,39.As described in 2.5, EPDs were developed to test the electrophoretic properties of particles. The performance of the EPD cell could be determined by testing the Commission International De L’Eclairage (CIE) Yxy chromaticity diagram. In the test, a square wave was selected as the driving waveform. Then, the edited driving waveform was imported into the function generator through a universal serial bus (USB). The voltage amplitude was amplified by a signal amplifier and then applied to the EPD. Next, brightness data was collected using a colorimeter and transmitted to the computer through a USB. Finally, the brightness variation curve data was recorded in real-time using admesy software. During the test, a square wave with a period of 10 s and a voltage of 5 V was generated by the function generator, which was amplified ten times by a voltage amplifier and connected to the EPD cell; this experiment tested three cycles in total.This measurement results of EPD brightness and chromaticity under the action of an applied electric field are shown in High ionization 1-butyl-1-methylpiperidinium bromide mono ionic liquid was successfully grafted onto the CuPc surface. The modified particles had good stability and high positive charges in the electrophoretic dispersion. Due to the existence of a mono ionic liquid, the Zeta potential of CuPc particles increased to +60.27 mV, which was improved by 97.61%. The blue CuPc particles had a reversible electrical response in the EPD cell with a bipolar voltage of ± 50 V. It was apparent that modifying particles with high ionization mono ionic liquid was expected to prepare color electrophoretic particles which have potential application in the field of material technology such as EPDs. Moreover, the results show that piperidine ionic liquid is a substitute for some surfactants and effective charge control agents because of its unique properties of high ionization degree in non-polar media.


Background: N7-methylguanosine (m7G) is an emerging research hotspot in the field of RNA methylation, and its role in tumor regulation is becoming increasingly recognized. However, its role in colorectal cancer (CRC) remains unclear. Hence, our study explored the role of m7G in CRC.Methods: The mRNA expression data and the corresponding clinical information of the patients with CRC were obtained from The Cancer Genome Atlas (TCGA). A m7G-related gene pair signature was established using the Cox and LASSO regression analyses. A series of in silico analyses based on the signature included analysis of prognosis, correlation analysis, immune-related analysis, and estimation of tumor mutational burden (TMB), microsatellite instability (MSI), and response to immunotherapy. A nomogram prediction model was then constructed.Results: In total, 2156 m7G-related gene pairs were screened based on 152 m7G-related genes. Then, a prognostic signature of seven gene pairs was constructed, and the patients were stratified into high- or low-risk groups. Better overall survival (OS), left-sided tumor, early stage, immune activity, and low proportion of MSI-low and MSI-high were all associated with a low risk score. High-risk patients had a higher TMB, and patients with a high TMB had a poor OS. Furthermore, the risk score was linked to immune checkpoint expression (including PD-L1), the tumor immune dysfunction and exclusion (TIDE) score, and chemotherapy sensitivity. We also created an accurate nomogram to increase the clinical applicability of the risk score.Conclusion: We identified an m7G pair-based prognostic signature associated with prognosis, immune landscape, immunotherapy, and chemotherapy in CRC. These findings could help us to better understand the role of m7G in CRC, as well as pave the path for novel methods to assess prognosis and design more effective individualized therapeutic strategies. Colorectal cancer (CRC) is the third most common tumor and the second leading cause of cancer-related deaths worldwide, with an estimated 1.8 million new cases and approximately 915,880 deaths worldwide in 2020 . With inin vivo , it is possible to systematically study the role of m7G regulators in CRC. Therefore, we developed a novel prognostic signature based on seven m7G-related gene pairs. We systematically and comprehensively investigated the potential role of the model in the clinical outcomes and tumor microenvironment (TME) of CRC. Our results provide additional data on prognostic biomarkers and therapeutic targets for CRC.https://portal.gdc.cancer.gov/). Patients without prognostic data were excluded from this study. In addition, data related to 562 patients were obtained from the Gene Expression Omnibus  databases. The TCGA dataset (training set) was employed to develop a prognostic signature, and the GSE39582 dataset , which contained data on 403 CRC samples, was used to validate the predictive accuracy of the signature. Additionally, data regarding 152 m7G regulators were obtained from a previous study  + (gene pair B coefficient × expression level) +…+ (gene pair n coefficient × expression level). Furthermore, according to the above formula, the risk scores of patients with CRC were calculated separately, and the patients were stratified into low- and high-risk groups based on the median risk score. Kaplan–Meier survival curves were plotted to estimate the OS differences between the two subgroups. A time-dependent ROC (ROC) curve was plotted to assess the accuracy of the signature.The external cohort GSE39582 was used to verify the m7G-related prognostic model. The risk score for each patient was calculated using the same coefficients and normalized expression microarray data for the m7G pairs. The patients were stratified into low- and high-risk groups based on the median risk score of the training set. Kaplan-Meier and tdROC curves were plotted to evaluate the prognostic value of the risk model.The associations between the risk score and clinicopathological traits  were compared. Univariate and multivariate analyses were performed to evaluate whether the risk scores were independent of the other clinical variables. Moreover, we conducted a stratified analysis to determine whether the risk score preserved its predictive capacity in the different subgroups.Based on the independent indicators, the R software “regplot” package was used to develop a nomogram for OS prediction at three and 5 years. Then, tdROC analysis for 3- and 5-year OS was performed to evaluate the prognostic accuracy, and the calibration curves were drawn to compare nomogram-predicted probability with actual survival.The fraction of immune cells in the two risk subgroups was assessed using the CIBERSORT algorithm. Differences between the low- and high-risk groups were detected using the Wilcoxon rank-sum test. We also used boxplots to evaluate the differences in the levels of expression of immunological checkpoints between the low- and high-score groups, which were retrieved from the literature. Moreover, we calculated the tumor immune dysfunction and exclusion (TIDE) score to predict the response to immunotherapy in CRC patients .The “maftools” R package was employed to reveal the mutation frequency in patients with CRC belonging to different risk subgroups. Subsequently, we analyzed the relationship between TMB and the risk scores. Using the Kaplan–Meier method, the OS rates derived from data pertaining to the CRC samples from the low- and high-TMB groups were compared. Next, we evaluated the synergistic effect of TMB and risk score on prognostic stratification. In addition, the association between the risk scores and MSI was explored.We explored the sensitivity of chemotherapy based on the gene expression levels using the “pRRophetic” R package . The cheThe differentially expressed genes (DEGs) were screened using the “limma” package in the R software by setting criteria with |log2  |> 1 and adjusted p-value < 0.05. Subsequently, the “Clusterfiler” R package was employed to perform the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.p < 0.05 unless otherwise stated.All analyses in this study were performed using the R software (version 4.0.3). Statistical significance was set at The m7G-related genes in the TCGA and GEO datasets were merged into an overlapping gene set, from which 135 m7G-related genes were shared in both sets. Pairwise comparisons were conducted using the algorithm described in the “Methods” section to calculate the risk score for each m7G-related gene pair for subsequent analysis. As a result, 2,156 m7G-related gene pairs were identified. We conducted a univariate Cox regression analysis on these pairs and selected 30 meaningful m7G pairs to generate the models . Next, LRisk score= (0.873933 × NCBP2.CCNB1 exp) + (0.974044 × DXO. RPS6KB1 exp) + (−1.08373 × YTHDF2. XPO1 exp) + (0.712567 × PIK3CA.NUDT7 exp) + (−0.60472 × MYC. EIF2S3 exp) + (−1.14792 × NUDT4. ATF5 exp) + (0.632646 × PML. IFIT5 exp).We calculated the risk scores of each sample in the training set and classified the patients into two subgroups (low- and high-risk). In both the training and validation sets, the risk distribution plot demonstrated that survival times increased with the risk score . The Kapp < 0.05, The relationship between risk score and clinicopathological factors was explored. We found that patients with right-sided CRC and advanced stage (stage III-IV) were associated with high-risk scores, whereas sex and tumor status showed no correlation with risk scores . FurtherTo generate personalized predictions for patients with CRC, we integrated the m7G score and clinicopathological parameters  to establish a nomogram scoring system . The preBased on the CIBERSORT algorithm, we noticed a decreased number of activated CD8 + T cells, M1 macrophages, and dendritic cells, as well as resting dendritic cells, and an increased number of M2 macrophages in the high-risk subgroup . The expp = 0.004; p = 0.019; p < 0.001; As illustrated in the waterfall charts, the mutated genes in the different risk groups were mainly APC, PIK3CA, SYNE1, KRAS, TTN, TP53, and MUC16, but the mutation rates of these genes were different in the high- and low-risk subgroups . TMB wasTo select potential drugs for patients with CRC, we investigated the correlation between risk scores and drug sensitivity using the “pRRophetic” R package. The IC50 values of doxorubicin, imatinib, JNK Inhibitor VIII, nilotinib, pazopanib, and thapsigargin in the high-risk subgroup were lower, whereas sorafenib and metformin had lower IC50 values in the low-risk subgroup . Taken tWe performed functional enrichment analyses to investigate the latent biological processes that affect risk scores. Based on the criteria of FC > 1.5 and adjusted p-value < 0.05, we screened 262 DEGs in the different risk groups. GO terms indicated enrichment in the collagen-containing extracellular matrix, extracellular matrix structural constituents, and external encapsulating structure organization . FurtherCRC is a highly heterogeneous disease, and the difference in heterogeneity provides a complex landscape for the prognosis of patients and their response to immunotherapy. The traditional prognostic evaluation system based on TNM staging has not been able to meet the requirements of precision medicine. Exploring the molecular mechanisms underlying CRC pathogenesis may provide key clues for identifying promising prognostic biomarkers or developing effective therapeutic targets. A growing number of studies have established that post-transcriptional RNA modifications play a significant role in modulating gene expression, as well as carcinogenesis and development, among which the most common modifications are m6a, m5c, m1a, and m7G . AccumulIn vitro experiments showed that KPT-330, an XPO1 inhibitor, inhibited cancer growth in a dose- and time-dependent manner. Similarly, XPO1 inhibition has been shown to enhance therapeutic response in CRC (In the present study, we developed a novel prognostic signature based on seven m7G gene pairs in patients with CRC using previously identified m7G-related genes with either higher or lower expression levels. Several m7G-related genes have been reported to be associated with CRC and other cancers. MYC is a master transcription regulator and is one of the most common oncoproteins associated with increased mortality . MYC acte in CRC . PIK3CA,e in CRC . It is oe in CRC . Additioe in CRC .+ T cell M1 macrophages, activated dendritic cells activated, and resting dendritic cells, in the low-risk group was obviously lower than that of the high-risk subgroup, while the infiltration level of M2 macrophages was increased in the high-risk score group. CD8+ T cell infiltration levels are usually associated with favorable clinical outcomes in most solid tumors (Infiltrating immune cells are the primary cells in tumor tissues and play vital roles in tumor biology . This std tumors . The lowd tumors . We assed tumors , and thed tumors . In thisd tumors . It is nd tumors . We founNevertheless, this study has some limitations. First, the signature was conducted based on public databases, which may have been influenced by inherent case selection bias. Second, clinical tissues and CRC cell lines should be employed to validate the expression levels of signature genes, and further investigation and experiments are needed to explore the detailed regulatory effects of m7G-related genes in CRC.We established a novel m7G pair-based risk signature based on seven m7G-related pairs in CRC. The risk score is closely related to the clinical features and prognosis of patients. The nomogram can serve as a counseling and clinical decision aid for clinicians. Tumor microenvironment analysis established a theoretical framework for future research on the connection between immunity and m7G-related genes in CRC.


C. elegans, recruitment of the Polo-like kinase PLK-2 to the synaptonemal complex triggers phosphorylation and inactivation of CHK-2, an early meiotic kinase required for pairing, synapsis, and double-strand break (DSB) induction. Inactivation of CHK-2 terminates DSB formation and enables CO designation and cell cycle progression. These findings illuminate how meiotic cells ensure CO formation and accurate chromosome segregation.Meiotic chromosome segregation relies on synapsis and crossover (CO) recombination between homologous chromosomes. These processes require multiple steps that are coordinated by the meiotic cell cycle and monitored by surveillance mechanisms. In diverse species, failures in chromosome synapsis can trigger a cell cycle delay and/or lead to apoptosis. How this key step in ‘homolog engagement’ is sensed and transduced by meiotic cells is unknown. Here we report that in  Most animals, plants, and fungi reproduce sexually, meaning that the genetic information from two parents combines during fertilization to produce offspring. This parental genetic information is carried within the reproductive cells in the form of chromosomes. Reproductive cells in the ovaries or testes first multiply through normal cell division, but then go through a unique type of cell division called meiosis. During meiosis, pairs of chromosomes – the two copies inherited from each parent – must find each other and physically line up from one end to the other. As they align side-by-side with their partners, chromosomes also go through a mixing process called recombination, during which regions of one chromosome cross over to the paired chromosome to exchange information. Scientists are still working to understand how this process of chromosome alignment and crossing-over is controlled.If chromosomes fail to line up or cross over during meiosis, eggs or sperm can end up with too many or too few chromosomes. If these faulty reproductive cells combine during fertilization this can lead to birth defects and developmental problems. To minimize this problem, reproductive cells have a quality control mechanism during meiosis called “crossover assurance”, which limits how often mistakes occur. C. elegans, whose meiotic processes are similar to those in humans. In C. elegans, a protein called CHK-2 regulates many of the early events during meiosis. During successful meiosis, CHK-2 is active for only a short amount of time. But if there are problems during recombination, CHK-2 stays active for longer and prevents the cell division from proceeding.Zhang et al. have investigated how cells can tell if their chromosomes have accomplished this as they undergo meiosis. They looked at egg cells of the roundworm Zhang et al. uncovered another protein that affects for how long CHK-2 stays switched on. When chromosomes align with their partners, a protein called PLK-2 sticks to other proteins at the interface between the aligned chromosomes. A combination of microscopy and test tube experiments showed that when PLK-2 is bound to this specific location, it can turn off CHK-2. However, if the chromosome alignment fails, PLK-2 is not activated to switch off CHK-2. Therefore, CHK-2 is only switched off when the chromosomes are properly aligned and move on to the next step in crossing-over, which then allows meiosis to proceed. Thus, PLK-2 and CHK-2 work together to detect errors and to slow down meiosis if necessary.Further experiments in mammalian reproductive cells will reveal how similar the crossover assurance mechanism is in different organisms. In the future, improved understanding of quality control during meiosis may eventually lead to improvements in assisted reproduction. Meiotic progression differs in many ways from a mitotic cell cycle. A single round of DNA replication at meiotic entry is followed by two nuclear divisions. Between replication and the first division is an extended period known as meiotic prophase, during which chromosomes pair, align through the process of synapsis, and recombine to establish physical links (chiasmata) between each pair of homologs. Together with sister chromatid cohesion, chiasmata direct bipolar orientation and segregation of homologous chromosomes during Meiosis I . SynapsiDuring early meiosis, double-strand breaks (DSBs) are induced and resected. These recombination intermediates undergo strand invasion to establish joint molecules between homologs, which recruit a set of factors collectively known as ‘pro-crossover’ or ‘ZMM’ proteins. Many of these intermediates eventually lose the ZMM proteins and are resolved as non-crossovers, while the subset that retains these factors is ‘designated’ to be resolved as crossovers (COs) .C. elegans, formation and initial processing of DSBs requires the activity of CHK-2, a meiosis-specific homolog of the DNA damage transducing kinase Chk2/CHEK2  (S. cerevisiae and S. pombe) and CHK-2 (C. elegans) kinases lack this regulatory domain. Activation is likely promoted by binding of these kinases to target motifs on other proteins, such as Hop1 in budding yeast  . Like alx-x-S/T) . While mx-x-S/T) , the meing yeast , resultise PLK-2 . Togethese PLK-2 . Thus, CC. elegans remain somewhat unclear.During early pachytene, recombination intermediates are established along chromosomes and can be detected as foci by immunofluorescence. By mid-pachytene, most of these recombination intermediates disappear, while six ‘designated crossover’ foci, which show strong localization of the cyclin homolog COSA-1 and other pro-CO factors, are detected in each nucleus, one per chromosome pair . This acDefects in synapsis or establishment of CO intermediates result in activation of a ‘crossover assurance checkpoint’ and a delay in cell cycle progression  signaling in proliferating cells revealed that the Polo-like kinase Plk1 enables mammalian cells to enter mitosis following DDR activation by phosphorylating the FHA domain of Chk2, which inhibits substrate binding . We wondC. elegans expresses multiple homologs of mammalian Plk1, including PLK-1, which is essential for mitosis and thus for viability, and PLK-2, which is dispensable for development but plays important roles in meiosis  that recruits Polo kinases to the SC  in which nuclei display bright COSA-1 foci, since this ratio is fairly consistent for a given genotype/condition, more so than the absolute length of this zone or number of nuclei. The ‘bright COSA-1 zone’ was reduced from 55% of prophase in wild-type hermaphrodites to 44% in plk-2 null mutants, 37% in PLK-2::AID+auxin, and 20% in syp-1(T452A)  . The delsynapsis . Depletisynapsis . SYP-1T4synapsis , but a msynapsis –4B, D. Tplk-2 null mutants, PLK-1 can partially substitute at Pairing Centers to promote pairing and synapsis . Thus, PLK-3 does not contribute to inactivating CHK-2, even in the absence of PLK-1 and PLK-2.Although mutation or depletion of PLK-2 or failure to recruit it to the SC delayed inactivation of CHK-2, markers for CHK-2 activity eventually disappeared and CO designation was detected in all these strains. We thus wondered how CHK-2 is inactivated in the absence of PLK-2. In synapsis . We testsynapsis . Comparesynapsis . Howeverreported , plk-3 mT452Asyp-1  plates seeded with OP50 bacteria at 20°C. All epitope- and degron-tagged alleles analyzed in this study were fully functional, as indicating by their ability to support normal meiosis and development  was expressed and purified as described previously (C. elegans cDNA library and cloned into pFastBac1 (Life Technologies) with a GST tag at its N-terminus. GST-PLK-2 was expressed in insect Sf9 cells using the standard Bac-to-Bac system (Life Technologies) and then purified using glutathione Sepharose (GE Life Sciences).Recombinant kinase-dead CHK-2  sample buffer. Proteins in SDS buffer were electrophoresed using gradient polyacrylamide gels . CHK-2 bands were excised and stored at 4°C. Proteins were then subjected to either in-solution or in-gel trypsin digestion, and phosphorylation sites were identified using mass spectrometry analyses (UC Davis).In vitro kinase assays were performed in a buffer comprised of 25 mM HEPES pH 7.4, 50 mM NaCl, 2 mM EGTA, 5 mM MgSOAn Xevo G2 QTof coupled to a nanoAcquity UPLC system  was used for phosphorylation site identification. Briefly, samples were loaded onto a C18 Waters Trizaic nanotile of 85 µm × 100 mm; 1.7 μm . The column temperature was set to 45°C with a flow rate of 0.45 ml/min. The mobile phase consisted of A (water containing 0.1% formic acid) and B (acetonitrile containing 0.1% formic acid). A linear gradient elution program was used: 0–40 min, 3–40% (B); 40–42 min, 40–85% (B); 42–46 min, 85% (B); 46–48 min, 85–3% (B); 48–60 min, 3% (B).m/z 556.2771 and introduced at 1 µl/min at 45-s intervals with a 3 scan average and mass window of ±0.5 Da. The Mse data were acquired using two scan functions, corresponding to low energy for function 1 and high energy for function 2. Function 1 had collision energy at 6 V and function 2 had a collision energy ramp of 18–42 V.Mass spectrometry data were recorded for 60 min for each run and controlled by MassLynx 4.2 . Acquisition mode was set to positive polarity under resolution mode. Mass range was set from 50 to 2000 Da. Capillary voltage was 3.5 kV, sampling cone at 25 V, and extraction cone at 2.5 V. Source temperature was held at 110°C. Cone gas was set to 25 l/hr, nano flow gas at 0.10 bar, and desolvation gas at 1200 l/hr. Leucine–enkephalin at 720 pmol/µl  was used as the lock mass ion at C. elegans and was downloaded from uniprot.org and then randomized. Searches were performed with trypsin specificity and allowed for two missed cleavages. Possible structure modifications included for consideration were methionine oxidation, carbamidomethylation of cysteine, deamidiation of asparagine or glutamine, dehydration of serine or threonine, and phosphorylation of serine, threonine, or tyrosine.RAW Mse files were processed using Protein Lynx Global Server (PLGS) version 3.0.3 . Processing parameters consisted of a low energy threshold set at 200.0 counts, an elevated energy threshold set at 25.0 counts, and an intensity threshold set at 1500 counts. The databank used corresponded to 2, 1 mM EGTA, 0.1% Triton X-100, 1 mM DTT, cOmplete protease inhibitors [Sigma #4693159001] and phosSTOP [Sigma #4906837001]). Lysates were processed using a Dounce homogenizer and sonicated using a Branson Digital Sonifier on ice and then centrifuged at 20,000 RCF for 25 min at 4°C. The supernatant was incubated with anti-ALFA selector  for 3 hr at 4°C. Beads were then washed six times with lysis buffer and three times with milli-Q water. Proteins on beads were then processed for phosphorylation site identification using mass spectrometry (UC Davis).To obtain synchronized young adults, five to six L4 larvae animals were picked onto standard 60-mm culture plate spread with OP50 bacteria. Animals were grown at 20°C for 5 days until starved. 90 plates of L1 larvae for each genotype or condition were washed into 1.5-l liquid culture supplemented with HB101 bacteria. Worms were then grown with aeration at 200 rpm at 20°C for 3 days to reach adulthood. Six hours prior to harvest, 1 mM auxin or 0.25% ethanol (solvent control) was added to the liquid culture. Harvested worms were frozen in liquid nitrogen and stored at −80°C. The frozen worms were then processed using a prechilled Retsch mixer mill to break the cuticle, thawed on ice in cold lysis buffer  at room temperature for 10 min at 10,000 × C. elegans were performed as previously described , transferred to membranes, and blotted with mouse anti-HA , Guinea pig anti-HTP-3 1:1500, , mouse aThe CHK-2-active zone was determined by immunofluorescence with an antibody recognizing phosphorylated HIM-8 and ZIM proteins . DesignaBright GFP::COSA-1 foci were identified using the intensity profile of GFP::COSA-1 throughout pachytene, as shown in Quantification methods and statistical parameters are described in the legend of each figure, including sample sizes, error calculations (standard deviation or standard error of the mean), statistical tests, and p values. p < 0.05 was considered to be significant. C. elegans. By revealing PLK-2-dependent and -independent mechanisms of CHK-2 activity, this work provides a valuable understanding of the major regulators of meiotic progression.Zhang et al. present convincing data describing a role for Polo-like kinase PLK-2 in restricting the activity of Chk2 kinase and coordinating synapsis of homologous chromosomes with the progression of meiotic prophase in  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Jeff Sekelsky (Reviewer #1).Thank you for submitting your article "Recruitment of Polo-like kinase couples synapsis to meiotic progression via inactivation of CHK-2" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The two main points highlighted below relate to the CHK-2 phospho-mutants and to the effect of the presence of the kinases as opposed to the requirement of their kinase activity. Most experiments we discussed presented complications and we therefore will not require them for publication. We would in turn 'suggest' them as a means to enhance the significance of the findings, if possible.1) Did you consider expressing the different CHK-2 mutants from heterologous loci, all in the background of endogenous AID::CHK-2? Alternatively, providing wild-type AID::CHK-2 from an alternative locus could be an option. We do appreciate that given the stability effect of the phospho-mutants, any of these strategies might not be helpful.2) Some attempts to differentiate between the requirement of the kinases vs the requirement of their kinase activity would be highly beneficial for the manuscript. Once again, given that these kinases play roles earlier in meiosis, the use of kinase-dead alleles might not be possible. Have the authors tried an analog-sensitive strategy to inhibit kinase activity?Additionally, please go over the reviewers' comments carefully as some of them point out that some claims might need rewording.Reviewer #1 (Recommendations for the authors):It is worth clarifying in the Results section how long it takes for a nucleus to progress through prophase I .1:4 – "CHK-2-dependent phosphorylation sites on HIM-8 and ZIM-1/2/3 at Pairing Centers" – I'm assuming this means this happens exclusively at pairing centers and not that the antibody only works at pairing centers, but the wording could imply the latter.11:1 – Typo ("paring" should be "pairing").Figure 1:The meaning of the axis label "% of bright GFP::COSA-1 zone" is unclear before carefully reading the figure legend. It would be best to additionally state what this metric represents in the actual body of the text.In Figure 1D, the bright green of GFP::COSA-1 in the graph is a bit challenging to read.Figure 2The color of SYP-1 (or at least the color of the text-the pseudocoloring of the microscopy appears to be magenta) should be changed to magenta to make the figure accessible to colorblind readers.Figure 2-S1:Change the color of RAD-51 to magenta.Figure 3-S1:The text size of the axes and labels of the mass spec graph could be increased to improve legibility.Figure 4-S1 and associated Methods text:Were the gain and/or brightness and contrast modified to account for the intense pHIM-8/ZIMs staining? If so, it would be informative to include this in the Microscopy methods.Reviewer #3 (Recommendations for the authors):Figure 2 legend: "Dashed green lines indicate the earliest nuclei with bright COSA-1 foci and the end of pachytene, respectively." – please clarify that the "respectively" refers to the left line and the right line.p7 lines 14,16: Is there a reference for high versus intermediate levels of CHK-2 activity in these backgrounds?  Otherwise, this should be marked as speculation.p. 9 line 5: The authors speculate that phosphorylation of CHK-2 T120 leads to CHK-2 degradation, which is plausible given their results; the unexpected finding that blocking this phosphorylation with mutation to alanine  also apparently leads to instability should be discussed.p. 9: the non-phosphorylatable alleles of CHK-2 turned out to be loss-of-function alleles, so the specific questions of whether phosphorylation of T120 or S116 leads to inactivation could not be asked in this work. Have the authors considered providing wild-type AID:chk-2 at a heterologous locus in these mutant backgrounds, to assess these alleles after depletion (after CHK-2 has performed its earlier function)?p. 10, lines 20-21: the quantitative difference in CHK-2 activity between the depletion of PLK-2 and SYP-1(T452A) mutants is striking and somewhat counterintuitive since PLK-2 presumably at least exists in the SYP-1 phosphomutant. Is it possible that the difference lies in wild-type SYP-1's ability to recruit PLK-1? This could be experimentally addressed or discussed.p. 12, line 19: The reference Sato-Carlton et al. 2020 shows that HIM-3 is phosphorylated independently of PLK-2 and PLK-1; this is opposite to what is stated here.p. 11, line 3: SYP-2 should be corrected to SYP-1.p. 17, line 4: correct "uniport" → "uniprot". Essential revisions:The two main points highlighted below relate to the CHK-2 phospho-mutants and to the effect of the presence of the kinases as opposed to the requirement of their kinase activity. Most experiments we discussed presented complications and we therefore will not require them for publication. We would in turn 'suggest' them as a means to enhance the significance of the findings, if possible.1) Did you consider expressing the different CHK-2 mutants from heterologous loci, all in the background of endogenous AID::CHK-2? Alternatively, providing wild-type AID::CHK-2 from an alternative locus could be an option. We do appreciate that given the stability effect of the phospho-mutants, any of these strategies might not be helpful.C. elegans. While methods for single-copy insertion  have enabled germline expression of transgenes, this often fails to recapitulate the expression levels of endogenous genes. This is usually attributed to the presence of multiple potent and silencing pathways (including RNAi and piRNAs) in the germline. It is particularly daunting to introduce chk-2 transgenes, since chk-2 is an unusually large gene for C. elegans (~8 kb), well beyond the typical size of insertions made with CRISPR/Cas9, MosSCI, or other available transgenic methods. Moreover, the endogenous chk-2 is on a chromosome arm; such distally positioned genes are typically protected from germline silencing by mechanisms that remain poorly understood, further complicating efforts to design and introduce additional copies. Thus, we feel that the effort required to overcome the technical barriers to such experiments outweighs any potential information that they would yield.We recognize that the instability we observed for CHK-2 mutant proteins makes their analysis less than fully satisfying. We considered various ways to circumvent this issue, including expressing these mutant alleles in the presence of a degradable wild-type copy (as suggested above). However, as the reviewers/editors have noted, such experiments can be technically challenging. One barrier is the challenges associated with expression of transgenes in the germline of 2) Some attempts to differentiate between the requirement of the kinases vs the requirement of their kinase activity would be highly beneficial for the manuscript. Once again, given that these kinases play roles earlier in meiosis, the use of kinase-dead alleles might not be possible. Have the authors tried an analog-sensitive strategy to inhibit kinase activity?C. elegans is severely limited by the low permeability of the worm cuticle and/or the activity of efflux pumps. We also found that CHK-2 is refractory to the analog-sensitive approach; our efforts to engineer such alleles resulted in severe loss of kinase activity in the absence of inhibitors.We appreciate this comment, as well as the reviewers’ recognition (again) that such experiments might be technically prohibitive. We agree that it would potentially be helpful to determine whether the role of PLK-2 and/or CHK-2 in promoting crossover designation can be separated from their kinase activities. We have engineered analog-sensitive alleles to inhibit PLK-1 and PLK-2 (as well as CDK-2) in other contexts, but this approach was not useful here for 2 reasons: (1) the degree of inhibition was somewhat variable, and more crucially, (2) the kinetics of inhibition were not rapid enough to allow for activity of the Polo-like kinases during early meiosis and inhibition shortly thereafter. The activity of most small molecules, including ATP analogs, in adult C. elegans.Auxin (indole acetic acid) is unusually small and water-soluble, and thus diffuses relatively quickly into the germline. It often induces robust protein degradation in ~1 hour, and this approach enabled us to induce degradation of CHK-2 and PLK-1/2 far more quickly than we could inhibit the kinase activity of PLK-1/2 using ATP analogs. Even this approach is limited by diffusion; we know that auxin-induced degradation can occur markedly faster in other experimental contexts, including in oocytes and embryos freshly dissected from adult Thus, we do not have suitable tools to address this issue beyond what we have already included in the manuscript.Additionally, please go over the reviewers' comments carefully as some of them point out that some claims might need rewording.Reviewer #1 (Recommendations for the authors):It is worth clarifying in the Results section how long it takes for a nucleus to progress through prophase I We have now addressed this in the figure legend of Figure 1A. Cells enter meiosis in the distal “arms” of the gonad and move proximally towards the spermatheca and uterus at a velocity of about one cell diameter per hour . This should be regarded as a rough approximation and varies over the reproductive lifespan of adult hermaphrodites.1:4 – "CHK-2-dependent phosphorylation sites on HIM-8 and ZIM-1/2/3 at Pairing Centers" – I'm assuming this means this happens exclusively at pairing centers and not that the antibody only works at pairing centers, but the wording could imply the latter.We thank the reviewer for pointing out this. We have revised the comment as follow:“CHK-2-dependent phosphorylation sites on the four paralogous pairing center proteins, HIM-8 and ZIM-1/2/3…”.11:1 – Typo ("paring" should be "pairing").Thanks for noting this; we have corrected the mistake.Figure 1:The meaning of the axis label "% of bright GFP::COSA-1 zone" is unclear before carefully reading the figure legend. It would be best to additionally state what this metric represents in the actual body of the text.We understand that for a ratio, it is more readable if both the numerator and the denominator are shown on a graph. But we feel it is not easy to precisely describe the denominator using simple words without causing confusion. So, we prefer just to indicate that the axis is a ratio on the graph and rely on the readers to read the figure legend if they need the detailed information.In Figure 1D, the bright green of GFP::COSA-1 in the graph is a bit challenging to read.We have changed the color to grey to improve readability.Figure 2The color of SYP-1 (or at least the color of the text-the pseudocoloring of the microscopy appears to be magenta) should be changed to magenta to make the figure accessible to colorblind readers.Thanks for noting this; we have made the suggested change.Figure 2-S1:Change the color of RAD-51 to magenta.We have changed the color of RAD-51 to magenta.Figure 3-S1:The text size of the axes and labels of the mass spec graph could be increased to improve legibility.We have made these changes.Figure 4-S1 and associated Methods text:Were the gain and/or brightness and contrast modified to account for the intense pHIM-8/ZIMs staining? If so, it would be informative to include this in the Microscopy methods.We thank the reviewer for this question. We think the reviewer is referring to the difference of pHIM-8/ZIMs intensity in the pre-meiotic nuclei in control and PLK-depleted worms. The intense signal in pre-meiotic nuclei does not correspond to HIM-8/ZIMs, but instead to an unknown epitope recognized by the antibody in mitotic cells . The intensity of this pre-meiotic epitope is greatly elevated by PLK-1/2 depletion, presumably due to arrest in mitosis. We adjusted the brightness in this figure to reveal the pHIM-8/ZIMs signal in meiotic nuclei, but the background intensity outside meiotic nuclei were kept the same in control and PLK-depleted worms, allowing comparison of signal intensity between different conditions. We have clarified this in the revised manuscript.Reviewer #3 (Recommendations for the authors):Figure 2 legend: "Dashed green lines indicate the earliest nuclei with bright COSA-1 foci and the end of pachytene, respectively." – please clarify that the "respectively" refers to the left line and the right line.We thank the review for this suggestion. We have changed the statement to “Dashed green lines on the left and right indicate the earliest nuclei with bright COSA-1 foci and the end of pachytene, respectively.”p7 lines 14,16: Is there a reference for high versus intermediate levels of CHK-2 activity in these backgrounds?  Otherwise, this should be marked as speculation.We have now elaborated our description of the differences seen in synapsis- versus DSB/recombination-deficient mutants. As reported in Kim et al. (2015) and elsewhere, synapsis defects prolong the clustered chromosome morphology (sometimes described as a “crescent-shaped” DAPI-staining mass) characteristic of the leptotene-zygotene stage, while defects in DSB induction or recombination result in an extended “early pachytene” stage, with chromosomes distributed around the nuclear periphery. More and brighter foci of phosphorylated HIM-8/ZIM proteins are seen in transition-zone like nuclei than in early pachytene nuclei, indicating that the level of CHK-2 activity, at least at the Pairing Centers, is higher in leptotene/zygotene than in early pachytene.p. 9 line 5: The authors speculate that phosphorylation of CHK-2 T120 leads to CHK-2 degradation, which is plausible given their results; the unexpected finding that blocking this phosphorylation with mutation to alanine  also apparently leads to instability should be discussed.We have discussed this in the revision.p. 9: the non-phosphorylatable alleles of CHK-2 turned out to be loss-of-function alleles, so the specific questions of whether phosphorylation of T120 or S116 leads to inactivation could not be asked in this work. Have the authors considered providing wild-type AID:chk-2 at a heterologous locus in these mutant backgrounds, to assess these alleles after depletion (after CHK-2 has performed its earlier function)?Please see our response to this suggestion above.p. 10, lines 20-21: the quantitative difference in CHK-2 activity between the depletion of PLK-2 and SYP-1(T452A) mutants is striking and somewhat counterintuitive since PLK-2 presumably at least exists in the SYP-1 phosphomutant. Is it possible that the difference lies in wild-type SYP-1's ability to recruit PLK-1? This could be experimentally addressed or discussed.Yes, we believe that phosphorylated SYP-1 may recruit a small amount of PLK-1, especially in the absence of PLK-2. We have discussed this in the revision.p. 12, line 19: The reference Sato-Carlton et al. 2020 shows that HIM-3 is phosphorylated independently of PLK-2 and PLK-1; this is opposite to what is stated here.We have corrected this mistake.p. 11, line 3: SYP-2 should be corrected to SYP-1.We have corrected SYP-2 to SYP-1.p. 17, line 4: correct "uniport" → "uniprot".We have corrected this typo in the revision.


The hypothalamus of the central nervous system is implicated in the development of diabetes due to its glucose-sensing function. Dysregulation of the hypothalamic glucose-sensing neurons leads to abnormal glucose metabolism. It has been described that fractalkine (FKN) is involved in the development of hypothalamic inflammation, which may be one of the underlying causes of a diabetic condition. Moreover, iron may play a role in the pathogenesis of diabetes via the regulation of hepcidin, the iron regulatory hormone synthesis. MicroRNAs (miRNAs) are short non-coding molecules working as key regulators of gene expression, usually by inhibiting translation. Hypothalamic miRNAs are supposed to have a role in the control of energy balance by acting as regulators of hypothalamic glucose metabolism via influencing translation.Using a miRNA array, we analysed the expression of diabetes, inflammation, and iron metabolism related miRNAs in the hypothalamus of a streptozotocin-induced rat type 1 diabetes model. Determination of the effect of miRNAs altered by STZ treatment on the target genes was carried out at protein level.We found 18 miRNAs with altered expression levels in the hypothalamus of the STZ-treated animals, which act as the regulators of mRNAs involved in glucose metabolism, pro-inflammatory cytokine synthesis, and iron homeostasis suggesting a link between these processes in diabetes. The alterations in the expression level of these miRNAs could modify hypothalamic glucose sensing, tolerance, uptake, and phosphorylation by affecting the stability of hexokinase-2, insulin receptor, leptin receptor, glucokinase, GLUT4, insulin-like growth factor receptor 1, and phosphoenolpyruvate carboxykinase mRNA molecules. Additional miRNAs were found to be altered resulting in the elevation of FKN protein. The miRNA, mRNA, and protein analyses of the diabetic hypothalamus revealed that the iron import, export, and iron storage were all influenced by miRNAs suggesting the disturbance of hypothalamic iron homeostasis.It can be supposed that glucose metabolism, inflammation, and iron homeostasis of the hypothalamus are linked via the altered expression of common miRNAs as well as the increased expression of FKN, which contribute to the imbalance of energy homeostasis, the synthesis of pro-inflammatory cytokines, and the iron accumulation of the hypothalamus. The results raise the possibility that FKN could be a potential target of new therapies targeting both inflammation and iron disturbances in diabetic conditions.The online version contains supplementary material available at 10.1186/s13098-022-00919-5. Diabetes, a systemic metabolic disease affects people all over the world. Diabetes can arise from different metabolic dysfunctions like insulin resistance, glucose intolerance, or from obesity . In receThe alterations of the neuron-glial cell interactions were also observed in the case of diet-induced metabolic changes and may contribute to the establishment of diabetes . FractalIron may play a role in the pathogenesis of diabetes. Iron overload of the human body increases the risk of the establishment of metabolic disorders , 12. RecThe molecular mechanisms with which the glucose metabolism, inflammatory processes, and iron homeostasis of hypothalamus crosstalk, are still under investigation. The role of small non-coding RNA molecules, the miRNAs have been investigated in metabolic disorders . The miRIn the present study, we investigated the molecular biological background and the possible links between diabetes, inflammation, and iron metabolism in the hypothalamus of an STZ-induced rat type 1 diabetes model.Using the miRNA array, we analysed the expression of diabetes, inflammation, and iron metabolism related miRNAs. Determination of the effect of miRNAs altered by STZ treatment on the target genes was carried out at protein level.Based on the findings, it is supposed that glucose metabolism, inflammation, and iron homeostasis of the hypothalamus are linked via the altered expression of common miRNAs as well as the increased expression of FKN, which contribute to the imbalance of energy homeostasis, the synthesis of pro-inflammatory cytokines and the iron accumulation and retention of the hypothalamus.Rattus norvegicus) were purchased from Janvier Labs . After one week of resting, the animals were randomly separated into two groups: Streptozotocin treated (n = 18) and control (n = 18). A total of 36 animals were used in the experiments. Streptozotocin  was solved in citrate buffer . The single dose of STZ was determined by body weight (65 mg/bwkg) and was injected i.v. into the tail vein for developing type 1 diabetes The control animals received a single i.v. injection of citrate buffer according to their body weight. Hyperglycaemia was determined by measuring the blood sugar concentration from the tail vein using the Accu-Chek blood sugar meter  after one week of injection. Rats were considered diabetic if the peripheral blood glucose concentration was higher than 20 mmol/L after one week of STZ treatment. Animals were kept under SPF conditions under permits BAI/35/90–5/2019, BA/35/77-2/2019, and BA/35/77-2/2020 (issued by the Baranya County Government Office). The Institutional Animal Care and Use Committee has approved the animal use protocols . All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. No mortality was observed and therefore no animals were excluded.Seven-week-old, male Wistar rats  and (∆∆Ct) method, the expression levels of miRNAs were determined as fold change compared to the untreated control. All kits were used according to the manufacturers’ protocols.Total miRNA was isolated from the hypothalamus tissue samples of 6 STZ-treated and 6 control animals using a miRPremier miRNA isolation kit . miRNA samples were pooled into STZ-treated and control groups. 250 ng of pooled miRNA of both groups was applied for cDNA synthesis using miScript II RT Kit . The mature miRNA expression profiling was performed with miScript miRNA PCR Assay for Rat diabetes using SYBR Green-based real time PCR protocol . miScript miRNA PCR Array data analysis was performed using Qiagen excel template for miScript miRNA PCR Array  using Aurum Total RNA Mini Kit . Complementary DNA was synthesised from 100 ng of total RNA using IScript Select cDNA Synthesis Kit  according to the manufacturer’s protocol. Determination of gene expressions was performed in a CFX96 One Touch Real-Time System  applying iTaq™ Universal SYBR® Green Supermix . The total reaction volume was 20 µL. The specificity of the reactions was determined by generating a melting curve after each run. Relative quantification was carried out by the Livak (∆∆Ct) method using the Bio-Rad CFX Maestro software 1.2. . β-actin housekeeping gene was used for normalization in each sample. The relative expression of the untreated samples was considered as 1. The mRNA expression of the STZ-treated samples was compared to the untreated samples and was expressed as fold change. The primer sequences used in this study are described in Table The hypothalamus tissue samples  were lysed in 150 µL of ice-cold lysis buffer  containing complete mini protease inhibitor cocktail . The protein contents of the samples were determined using a DC Protein Assay Kit . Protein samples were separated into 10% or 12% polyacrylamide gels. The gels were transferred by electroblotting to nitrocellulose membranes . The following polyclonal rabbit antibodies were used for WB experiments: anti-GLUT4 IgG , anti-insulin receptor (INSR) IgG , anti-hexokinase 2 (HXK-2) IgG , anti-glucokinase (GCK) IgG , anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) IgG , anti-leptin receptor IgG , anti-fractalkine (CX3CL1) IgG , anti-fractalkine receptor (CX3CR1) IgG , anti-ferroportin (Fp) IgG , anti-divalent metal transporter 1 (DMT-1) IgG  anti-transferrin receptor 1 (TfR1) IgG , anti-ferritin light (FTL) chain IgG , anti-mitochondrial ferritin (FTMT) IgG , anti-ferrochelatase (FECH) IgG , anti-NFS-1 IgG . All these primary antibodies were used for 1 h incubation at room temperature. Ferritin heavy chain (FTH) IgG  was used overnight at 4 °C. Anti-β-actin IgG  was used as loading control of the Western blots. Horseradish peroxidase (HRP)-linked goat anti-rabbit IgG was used  as the secondary antibody, for 1 h at room temperature. Western blot development was performed with traditional colourimetric detection using Fuji medical X-ray film  and WesternBright ECL chemiluminescent substrate . Optical densities of the protein bands were analyzed by ImageJ software . The resp-value < 0.05.The miRNA PCR assay was performed in triplicate. Real time PCR determinations were carried out in triplicate in three independent experiments. Western blots are representative of three independent experiments. Statistical analysis was performed using SPSS software . Statistical significance was determined by the Student’s t-test. Data are shown as mean ± standard deviation (SD). The results were considered statistically significant at a diabetes mellitus. On the other hand, the relative mRNA expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme contributing to diabetic symptoms significantly decreased to 0.53 ± 0.06 in the STZ-treated rat hypothalamus compared to the control mRNA level, which was regarded as 1.One week after the treatments, the blood sugar concentrations of the animals in the control group and the STZ group were 6.95 ± 1.05 mmol/L (n = 18), and 27.09 ± 5.29 mmol/L (n = 18), respectively showing the development of hyperglycaemia. The body weight of the control animals was 260 ± 10.14 g at the beginning of the experiment and 311.21 ± 12.25 g at the end of the experiment. The body weight of the animals was 258 ± 11.2 g before the STZ treatment and 272 ± 11.81 g after one weak STZ treatment showing a reduced rate of body weight gain. The relative mRNA expression level of phosphoenolpyruvate carboxykinase (PEPCK) was significantly increased in the hypothalamus of STZ-treated animals to 14.58 (± 2.52) compared to the expression level of the control, which was considered as 1, showing disturbance in the gluconeogenesis, which is a sign of The miRNAs regulating glucose metabolism show changes in the STZ-treated rat hypothalamus and modify the protein levels of the target genes.We examined the relative expression of 88 rno-miRNAs with miRNA PCR Assay. According to the significance of clinical aspects, 18 miRNAs were chosen for further investigation. The miRNAs related to glucose metabolism showing alteration in the expression rate at STZ treatment are listed in Table The levels of GLUT4 and HXK-2 translational regulators rno-miR-135b-5p and rno-miR-21-5p were increased, as well as the mRNA levels of GLUT4 glucose transporter and HXK-2, which resulted in the upregulation of the levels of both proteins. However, the expression of INSR regulatory miRNAs rno-miR-194-5p and rno-miR-200a-3p altered in an opposite direction, and the mRNA level of INSR did not change significantly compared to the control, the protein expression of INSR decreased. The decreasing level of rno-miR-200a-3p and the increasing level of LEPR mRNA eventuated to significantly elevated LEPR protein expression. Both the GCK regulator rno-miR-152-3p and the IGFR1 regulator rno-miR-96-5p showed decreased levels. Meanwhile, the elevation of GCK and IGFR1 mRNA expression was observed in the hypothalamus of the STZ-treated animals compared to the controls. Finally, the elevated level of rno-miR-29a-3p showed a positive correlation with the mRNA expression level of PEPCK  cycle , 80.Based on our findings the STZ-induced diabetic condition affects hypothalamic glucose metabolism, inflammation, and iron homeostasis. The underlying molecular mechanisms mediating interactions between these processes are incompletely understood. It is supposed that they are linked together via the altered expression of common miRNAs as well as the increased expression of FKN. FKN acts as a regulator of inflammation and iron metabolism via controlling the downstream signalling cascades mediated by FKN/CX3CR1 interaction and it appears to work as a link between diabetes, inflammation, and iron metabolism. The dysregulation of hypothalamic iron homeostasis may contribute to the degeneration of neuronal cells and the imbalance of energy homeostasis. Our results raise the possibility that FKN could be a potential target of new therapies targeting both inflammation and iron disturbances in diabetic conditions.Additional file 1: Expression level of glucose metabolism-related miRNAs and mRNA analysis of the target genes in the liver of STZ-treated animals.


COVID-19 increases the risk of acute ischemic stroke. The objective of this study is to identify the risk factors, comorbidities, and outcomes in COVID-19 patients with acute ischemic stroke at University of Indonesia Hospital.p < 0.05), patients at the critical COVID-19 clinical stage, and patients with atrial fibrillation (p < 0.05). The level of C-reactive protein (CRP) more than 60 mg/L correlated with the outcome of the patient as well.The clinical condition of COVID-19 patients with ischemic stroke is more severe for patients older than 55 years (The study concluded that, age, COVID-19 clinical degree, and atrial fibrillation significantly affect the outcome in COVID-19 patients with stroke. Corona virus disease 2019 (COVID-19) infection is a dangerous infection because of its ability to induce multiorgan failure in humans. Aside from the respiratory system, it may affect the cerebrovascular system, which is manifested as a stroke that occurred in that patient. Stroke is one of the neurological diseases that have high morbidity and mortality. Moreover, treating this condition creates a high economic burden [d-dimer level, erythrocyte sedimentation rate, and lactic acid dehydrogenase enzyme, and the decrease in lymphocyte. In addition to, the elevation of the liver enzyme and kidney biomarkers which suspected to play roles in those patients [The COVID-19 infection increases the risk of ischemic stroke occurrence by several mechanisms, including, activation of coagulation and inflammatory factors reflected on the increased patients .The severity of COVID-19 infection detected clinically based on the guidelines of the Decree of the Minister of Health of the Republic of Indonesia Number Hk.01.07/Menkes/4641/2021 concerning the Control of COVID-19. The mild degree of the COVID-19 is symptomatic without dyspnea, and the severe-critical degree is acute respiratory distress syndrome (ARDS) with oxygen saturation < 93%, sepsis and septic shock [In Indonesia, University of Indonesia Hospital had reported the cases of stroke in COVID-19 patients without vascular comorbid. However, a greater amount of data was required to understand better the risk factors in COVID-19 that increased the risk of ischemic stroke. Finally, understanding the pathophysiology of stroke in COVID-19 patients and the mechanisms of each risk factor was essential as consideration for COVID-19 therapies and the prognosis of stroke in COVID-19 patients.d-dimer, C-reactive protein (CRP), and procalcitonin. D-dimer was measured by using FREND System manufactured in South Korea by NanoEntek company while CRP test was carried out by using Epithod 616 machine that was manufactured in South Korea by DxGen Corp. Procalcitonin test was measured by RAMP System manufactured in Canada by Response Medical Corp. The collected data were analyzed using the SPSS-26 application produced by IBM in Chicago in April 2019 and by comparing risk factors and the comorbidities of patients we may identify the dominant factors which play more roles in causing the stroke.This study was a cross-sectional study where patients’ electronic medical records were tracked from March 2020 to August 2021. The inclusion criteria for these patients were the hospitalized patients with positive COVID-19 infection identified by real-time PCR tests that stated the clinical symptoms of stroke and were supported by brain CT scans and MRI procedures. There were 52 stroke patients selected from a total of 26,154 hospitalized COVID-19 patients. The risk factors of the patients which were analyzed include age, gender, stroke types, hypertension, diabetes, hyperlipidemia, atrial fibrillation, chronic kidney disease, coronary artery disease, thorax radiology, COVID-19 severity degree, inflammatory markers such as d-dimer and CRP, while procalcitonin level increased in about half of the patients (51.1%) (Table The majority of the patients (82.7%) were older than 55 years old. Male numbers 61.5%) dominated the number of female patients (38.5%). The ischemic stroke was reported in 96.2% of patients, and hypertension (69.2%) was the most frequent risk factor followed by atrial fibrillation (19.2%). Most of the patients  demonstrated an elevation in .5% dominOut of 52 patients, there were 49 patients under cover of anticoagulant drugs as, 43 patients were treated with heparin (82.7%), five patients (9.6%) were treated with LMWH, and only one patient 1.9%) under oral anticoagulant . The systolic and diastolic pressure averages were 140.50 mmHg and 79.58 mmHg, respectively. The MAP was 100.85 mmHg. The leukocyte count average was 14.07 × 103/µL, lymphocyte was 14.07, and neutrophil was 80.84%. The erythrocyte count sedimentation average rate was 58.75 mm. The urea average level was 87.15 mg/dL. Then, the creatinine level was 2.03. The SGOT and SGPT level were 70.46 U/L and 49.61 U/L, respectively. Next, the HbA1C was 6.43. The There were 43 out of 52 patients older than 55 years . This could be due to the fact that atrial fibrillation was one of the independent ischemic stroke risk factors [Atrial fibrillation was another condition that worsened the outcome of stroke patients with COVID-19 infection. A study conducted by Qureshi and his colleagues  indicate factors .p: 0.00) as severe or critical COVID-19 conditions increased the patients' mortality by 40 times . This is supported by a study conducted by Vidale and his colleagues [This study also reported that the more severe clinical conditions of COVID-19 infection significantly affected the outcome of stroke patients with the higher mortality rate compared to stroke patients with moderate COVID-19 infection (lleagues  that thelleagues . Fridmanlleagues  as well lleagues .d-dimer level for survived patients was 353 ng/mL (μg/L), which increased to 529 ng/mL after 1 week compared to patients who passed away with a higher initial point of d-dimer at 869 ng/mL and 1494 ng/mL at the end of the week [d-dimer, however, a significant cutoff was not identified. This finding differed from the study carried out by Tang and his colleagues [d-dimer level in COVID-19 patients was closely responsible for poor prognosis and higher mortality rate [d-dimer level and mortality outcome. The high d-dimer was suspected due to the inflammation in COVID-19 infection, which leads to the coagulation cascade. Therefore, the significance of mortality etiology was still inconclusive.Based on a study carried out by Lodigiani and his colleagues , the medthe week . In thislleagues , which iity rate . This cop: 0,08). This could be an important finding which differed from other studies considering a meta-analysis study conducted by Yassin and his colleagues [C-reactive protein or CRP serum was discovered to be an essential marker, which might change significantly in severe COVID-19 patients. CRP was a protein produced by the liver, which had a role as an initial marker of infection and inflammation. In this study, the elevated CRP was observed in 86% of severe COVID-19 patients and more than 60 mg/L of CRP was reported as a cut-off point and thrice tendency for higher mortality   [Fridman and his colleagues  stated t4–5.855) , it was 4–5.855) . Meanwhi4–5.855) , 58 out Age of the patients significantly affected the outcome in stroke patients with COVID-19, where the older the patients age, atrial fibrillation, and the more severe the COVID-19 infection, associated with worse outcome, and the higher the chance of mortality. The study also suggested that CRP of higher than 60 mg/L played an important role. Further studies with a higher sample number are required to better understand the pathophysiology behind these findings.


Same-day surgery cancellation results in decreased operating room (OR) utilization, reduced productivity, and inconvenience for patients. We aim to assess the cancellation rates of elective surgeries, identify common causes, and evaluate changes due to the COVID pandemic.A retrospective cohort study was conducted identifying all same-day surgery cancellations at a tertiary pediatric academic hospital from 1/1/2015 to 12/31/2017 (pre-COVID) and from 4/1/2020 to 3/31/2021 (post-COVID). Statistical analysis was performed using generalized regression with cancellation as the dependent variable. Period, age, ethnicity, gender, preferred language, and insurance were independent variables.There were 55465 scheduled cases (41670 before and 13795 after COVID), with 1508 cancellations (2.7%). Of those, 1247 (3.0%) were before COVID and 261 (1.9%) after COVID (p < .001). Of all cases, 56.7% (31475) were male, 55.1% (30595) were non-Hispanic/Latinx whites, 82.3% (45638) spoke English, and 45.5% (25237) had public insurance. The mean age was 8.5 years (SD = 6.03).The decrease in the probability of cancellation was most significant in patients with public insurance, < 1 year-of-age, Hispanic/Latinx who spoke Spanish .There was a significant decrease in same-day cancellations post-pandemic. We hypothesize that the required pre-operative COVID test helped to minimize same-day cancellations. Increased communication and education enhanced family engagement and was critical for improved OR metrics, including cancellation rates.level IV. CanceThe aim of this study is to assess the cancellation rates of elective surgeries performed at a tertiary pediatric academic hospital, identify common causes and evaluate changes brought on by the COVID-19 pandemic.2A retrospective cohort study was conducted examining all patients who underwent elective procedures as well as those who cancelled the elective procedures on the same-day of the surgery from 1/1/2015 to 12/31/2017 (pre-COVID) and from 4/1/2020 to 3/31/2021 (post-COVID) at Rady Children's hospital, a 511-bed free-standing academic pediatric tertiary care hospital in San Diego, California. This study was reviewed and approved by the institutional review board (IRB) of Rady Children's hospital and University of California, San Diego (study # 181752). Data extraction from the electronic medical record (EMR) system used reporting work bench tool embedded in the EMR without retaining individual protected health information. Eligible cases were defined as elective cases that were on the published OR schedule by the end of the previous business day (5pm) before surgery and included only cases where patients were coming from home (outpatient or same-day admission). Any inpatient, trauma, urgent or emergent add-on cases were excluded from the analysis. This project was initiated well before the onset of the COVID-19 pandemic in order to analyze OR efficiency. Therefore, we used “baseline” data from 1/1/2015 to 12/31/2017 as “pre-pandemic.” After the onset of the COVID-19 pandemic, we obtained approval with an amendment to the study through the IRB to include the data from 4/1/2020 to 3/31/2021 and designated this period as “post-pandemic.”Statistical analysis was performed using JMP Version 14 , with two-sided p-values less than 0.05 considered to be statistically significant. Age differences between pre- and post-pandemic as well as comparison of the total cases with cancelled cases were analyzed using two-sample t-tests. A generalized regression with cancellation as the dependent variable was created. Period, age, ethnicity, gender, preferred language, and insurance were independent variables. A prediction profiler was used to optimize the generalized regression model.https://www.researchregistry.com/browse-the-registry#home/registrationdetails/62ac04f37dc393001ef2e571/.The study is complaint with the STROCSS 2021 criteria for reporting of cohort studies in surgery , and is 3There was a total of 55465 scheduled cases (41670 before and 13795 after COVID), with 1508 cancellations, an overall rate of 2.7%. The cancellation rate during the pre-pandemic period was 3.0% (1247 cases); and the cancellation rate was significantly lower post-pandemic at 1.9% (261 cases) (p < .001). Of all cases, 56.7% (31475) were male, 55.1% (30595) were non-Hispanic/Latinx whites, which is defined as Americans who identify themselves as “white” and are not of Hispanic or Latino heritage according to the United States Census Bureau [During the pre-pandemic period, there was a total of 41670 cases with 3.0% cancellation rate . There wInterestingly, the COVID pandemic has positively impacted the OR utilization with significantly lower rate of cancellation compared to our baseline. Regardless of the period, patients who had public insurance and those of Hispanic ethnicity had a higher rate of surgery cancellations (p < .001). By optimizing the factors in the generalized model profiler we found that the decrease in the probability of cancellation rate was most significant in patients who had public insurance, younger than 1 year-of-age, were Hispanic/Latinx and spoke Spanish , and those with same-day cancellation were significantly younger with a mean age of 7.3 (SD 6.1) (p < .001). The mean age of the post-pandemic cohort was significantly older at 9.3 (SD 6.2) when compared to the pre-pandemic cohort (p < .001), and those with same-day cancellation had a slightly younger mean age of 8.6 (SD 6.3) which was not statistically significant (p = .07). During the pre-pandemic period, the average scheduled case length for cancelled surgery was 80 min (SD = 62). During the post-pandemic period, the average scheduled case length for cancelled surgery was 90 min (SD = 74). In surgery, estimates of the cost of OR time vary widely without well-established benchmarks. In an effort to understand costs of care, Childers et al. calculated the mean cost of OR time at $36 to $37 per minute, using financial data from California's short-term general and specialty hospitals in FY2014 [When analyzing reasons for cancellation, we separated the reasons into patient-related and non-patient-related categories . During 4The same-day elective surgical case cancellation rate reported during baseline pre-pandemic period was quite low compared to many published reports , and thiWe found that both Hispanic ethnicity and public insurance status were associated with increased probability of same-day cancellation. This likely reflects a degree of disparity in care, and private insurance has been found to be associated with decreased rate of cancellation even in countries with universal healthcare. The othAfter the onset of the COVID-19 pandemic, there was a significant change in pre-operative workflow regarding scheduling of elective surgical cases. At our institution, COVID-19 RT-PCR testing became a requirement very early on during the pandemic in order to keep the healthcare staff and patients safe. We stipulate that the mandatory pre-operative COVID testing required by the hospital 48 h prior to elective surgery helped minimize same-day cancellations. Although inconvenient for the family, the requirement of coming to the healthcare facility for the COVID test 2 days prior to surgery helped to reduce the “no show” rate since it is intuitive that if family already committed time and energy to come for the pre-operative testing, they are more likely to come on the day of the scheduled procedure. The extra visit could also allow the family to become acquainted pre-operatively with the facility  as well as allowing them to have another opportunity to clarify any pending questions before the procedure. This is reflected in the decreased cancellation rates related to the categories of “no show” and “family refusal.” Since the pre-operative COVID tests were all scheduled over the phone with verification of time and date of the surgery, the increased nursing communications  and educIt has been well-documented that since the onset of COVID-19, due to increased mask mandates and social distancing, there has been a significant reduction in respiratory illnesses in the pediatric population due to other viral pathogens . At the Not surprisingly, in the post-pandemic period, cancellation due to OR scheduling conflicts was significantly higher (0.29%) when compared to the pre-pandemic period (0.08%). This is likely due to periodic staffing shortage with “unscheduled time off” during COVID surges where the rates of the COVID-19 infection in the county were high, decreasing available OR block time.The current study has a number of limitations. Given the retrospective nature of the study, documentation of the cancellation reasons may not always be accurate, and reasons may have been miss-classified. There may be other confounders that contributed to the cancellation rates that were not extracted or analyzed from the EMR. While many factors are significantly associated with increased risk of cancellation, the data presented here does not establish causality. Given our geographic location at the border between the United State and Mexico, the large proportion of Hispanic patients represented in our study mirrored the population composition of the San Diego county. Therefore, the demographic results reported here may not be generalizable to other geographic areas of the US. Given the variable mix of pediatric surgical subspecialties at individual children's hospitals, the reasons for cancellation due to both patient and surgeon-related causes may be different at another center since many of the reasons may be specialty dependent. Lastly, the data presented may not be easily extrapolated to other adult, non-tertiary, and international hospitals due to different surgical patient populations. However, the message that better patient education and communication are essential in improved care delivery is applicable in all healthcare settings.The analysis gave us insights to the reasons for same-day cancellation. Although the overall rates reported here is lower compared to previously published data, we believe that there is still room for improvement as demonstrated by the decreased rate after the onset of the COVID-19 pandemic, especially in categories of patient-related reasons that are preventable while cancellation rate due to non-preventable reasons remained stable.5The COVID-19 pandemic has adversely impacted our entire healthcare system in the US since its onset in 2020. Although it has caused many inconveniences for both patients and providers, it did somehow have a positive impact in lowering our same-day elective surgical cancellation rate. We found that regardless of period, certain populations are vulnerable with increased risk of cancellation such has Hispanic ethnicity and public insurance status.Once COVID infection rates decrease in the general population, we anticipate that pre-operative COVID testing may not be required for routine elective surgical procedures in the future. We would like to maintain the lower cancellation rate with the anticipated changes in workflow. Some of this may not be possible as we are already seeing increased rate of seasonal non-COVID viral upper respiratory infections especially in young children during 2021–22 season compared to the same period in 2020–2021 . HoweverThis study was reviewed and approved by the institutional review board (IRB) of Rady Children's hospital and University of California, San Diego (study # 181752).There is no funding source for this study.Study conception and design: Wen Jiang and Daniela Carvalho.Data acquisition: Wen Jiang.Analysis and data interpretation: Daniela Carvalho.Drafting of the manuscript: Wen Jiang.Critical revision: Daniela Carvalho.Not applicable for the current study.1.researchregistry.comName of the registry: 2.Unique Identifying number or registration ID: researchregistry80153.https://www.researchregistry.com/register-now#user-researchregistry/registerresearchdetails/62ac04f37dc393001ef2e571/Hyperlink to your specific registration (must be publicly accessible and will be checked): None.Not commissioned, externally peer-reviewed.None of the authors have anything to disclose.


All the samples, without and with the alkaline process, were characterized for their physico-chemical, microstructure, thermal properties and compared to commercial cellulose (COM-C). The extraction yield was the highest in alkaline-extracted cellulose from the corncob (AE-CCC) sample (p < 0.05), compared to the other alkaline-treated samples. The undesired components, including mineral, lignin, and hemicellulose, were lowest in the AE-CCC sample (p < 0.05), compared to raw and alkaline-treated samples. The microstructure displayed the flaky AE-CCC structure that showed a similar visibility in terms of morphology with that of the alkaline-treated pineapple peel cellulose (AE-PPC) and COM-C samples compared to other alkaline-treated samples with a fibrous structure. Fourier Transform Infrared (FTIR) and X-ray Diffraction (XRD) of AE-CCC samples showed the lowest amorphous regions, possibly due to the elimination of hemicellulose and lignin during bleaching and alkaline treatment. The highest crystallinity index obtained in the AE-CCC sample showed a close resemblance with the COM-C sample. Additionally, the AE-CCC sample showed the highest thermal stability, as evidenced by its higher Tonset (334.64 °C), and Tmax (364.67 °C) compared to the COM-C and alkaline-treated samples. Therefore, agricultural wastes after harvesting in the Chiang Rai province of Thailand may be subjected to an alkaline process with a prior bleaching process to yield a higher cellulose content that is free of impurities. Thus, the extracted cellulose could be used as an efficient, eco-friendly, and biodegradable material for packaging applications.Cellulose is an abundant component of the plant biomass in agricultural waste valorization that may be exploited to mitigate the excessive use of synthetic non-biodegradable materials. This work aimed to investigate the cellulose utilized by alkaline extraction with a prior bleaching process from rice straw, corncob, Phulae pineapple leaves, and Phulae pineapple peels. The bleaching and alkaline extraction process was performed using 1.4% acidified sodium chlorite (NaClO Synthetic materials are employed to produce the packaging materials obtained from petroleum-based by-products that, after a single use, are disposed of in the environment . The appAnanas comosus L.) Merr., popularly known as “Phulae” can be only grown in certain parts due to its compatibility with the geographical location and climatic conditions of Chiang Rai, Thailand [The agriculture sector accounts for about 46% of the total land production area in Thailand . As a reThailand . The PhuIncreased agricultural production generates a bulk quantity of agricultural wastes after crop harvesting in the fields. A significant quantity of these wastes remains underutilized in the form of rice straw, pineapple leaves, and corn cobs in the harvesting fields of northern Thailand. Eventually, most of these wastes are burnt by the local farmers in an open space, generating a large quantity of smoke that severely impacts human health and the environment. Most farmers are unaware of the value of waste recycling and its economic potential due to lack of knowledge . The maj2) and hydrogen peroxide (H2O2) to remove all the residual lignin, wax, and lipids [The extraction of cellulose from agricultural wastes is subjected to pre-treatments using toluene, ethanol, or petroleum ether to remove the lipids, wax, phenolic, and pigments ,16. Afted lipids . However2H6O, 95%) were attained from RCI Labscan Co., Ltd. . Sodium chlorite  was bought from Ajax Finechem Pty Ltd. . Acetic acid glacial  was purchased from QRëC™ . All the materials and chemicals used in our study were of analytical grade.Commercial cellulose from cotton was purchased from Chanjao Longevity Co., Ltd. . Potassium hydroxide  was procured from Sigma Aldrich Co. . Toluene (99.8%) and ethanol (COryza sativa var. glutinosa), corncob (CC) (Zea mays var. indentata), Phulae pineapple leaves (PL) (Ananas comosus var. Phulae), and Phulae pineapple peels (PP) (Ananas comosus var. Phulae) were collected from the agricultural fields of Thasud, Chiang Rai, Thailand in the month of September 2021. RS, CC, PL, and PP raw materials with no apparent damage were collected and transported to the Department of Food Technology, Mae Fah Luang University, Chiang Rai, Thailand. All the samples were washed with distilled water to remove dust and adhered soils particles. After the washing process, all the samples were cut into 3–5-cm pieces and dried in a hot air oven at 60 °C for 48 h. The dried samples were ground, and 30 g of each RS, CC, PL, and PP samples were dissolved in 450 mL of toluene: ethanol (2:1) and left for shaking in a closed cabinet of bench-top temperature-controlled orbital shaker  at 150 rpm and 25 °C for 20 h. All the samples collected after the shaking process were filtered through Whatman No. 4 filter paper  using a Buchner funnel equipped with a vacuum pump. After removal of the solvent by filtration, all the samples were washed with absolute ethanol and filtered again before drying for 1 h at 100 °C. Dried fibrous matter of all the samples was mixed with 1.4% (w/v) sodium chlorite (NaClO2) to bleach the sample fibers. The pH of all the samples was adjusted to 4 by using 5% (v/v) acetic acid solution followed by heating at 70 °C with continuous stirring using an overhead stirrer at 500 rpm for 5 h. The sample fibers were filtered and washed with distilled water until a neutral pH was maintained and dried at 100 °C for 16 h. After bleaching, dried fibers were soaked in 600 mL of 5% (w/v) potassium hydroxide (KOH) and stirring was continued at 500 rpm under room temperature for 24 h prior to heating at 90 °C for 2 h for the extraction of cellulose. The KOH-treated samples were washed with distilled water until a neutral pH was attained, followed by drying in a hot-air oven at 100 °C for 20 h to obtain cellulose [Rice straws (RS)  and the dried mass of bagasse (g) using the Equation (1), as follows .Yield . The lignin contents of all the samples were measured using T222 om-98 method, and holocellulose contents (α-cellulose and hemicellulose) were estimated using the acid-chlorite method .A field emission scanning electron microscope (FESEM)  was employed to examine the microstructure of extracted cellulose samples. The samples were mounted on double-sided carbon tape, vacuum-dried, sputtered with gold, then scanned at a 3 kV accelerating voltage and 5000× magnification .w/w). The spectra were obtained in transmittance mode from a total scan of 32 scans with a resolution of 4 cm−1 over the 4000–400-cm−1 range.FTIR spectra of raw materials and cellulose samples were analyzed using an FTIR spectrophotometer  to characterize the functional groups of the samples . SamplesXRD patterns of fiber and cellulose were examined using an XRD diffractometer . The machine was operated at 40 kV and 30 mA, equipped with Cu Kα radiation at a wavelength of 1.54056 Å using a nickel monochromator filtering wave. The samples were scanned at room temperature in a range of 2θ = 5–40° at a scanning rate of 0.4°/min. The percentage of crystallinity index (CI) was calculated following Equation (2):2 gas atmosphere, in which all the samples were heated from 25 °C to 600 °C at a heating rate of 20 °C/min and a gas flow rate of 60 mL/min. For TGA, Derivative Thermogravimetry (DTG) curves were used to calculate the onset (Tonset) and maximum decomposition temperature (Tmax) of samples, while TGA curves were used to determine the char residue at 600 °C (%).The TGA of extracted cellulose was assessed through a thermogravimetric analyzer , following the conditions of Rashid and Dutta . Approxip < 0.05. Three replications were carried out for extraction yield and Chemical composition and one replication for SEM, FTIR, XRD and thermal properties.Analysis of variance (ANOVA) and Duncan’s multiple range test were performed using a statistical program, SPSS  version 26. Samples were analyzed at a significance level of w/w), respectively. The highest extraction yield was attained in alkaline-extracted cellulose from corncob (AE-CCC) compared to the other samples with alkaline treatment (p < 0.05). The extraction yield was related to the α-cellulose content in raw materials. The ash content of alkaline-treated samples was lower than that of raw material samples (p < 0.05). The lower ash content might be due to the removal of minerals during bleaching and repeated washing cycles throughout the extraction process. The chemical components obtained in RS, CC, PL and PP samples were in the range of 33.18–45.81% for α-cellulose, 27.88–44.15% for hemicellulose, and 12.70–27.25% for lignin. In comparison to all the raw material samples, the CC sample showed the highest α-cellulose content (45.81%) (p < 0.05), followed by RS (45.45%), PL (35.35%), and PP (33.18%), respectively. The results were in line with the α-cellulose content of straw from cereal crops and fresh fruit by-product residues [p < 0.05). Due to the removal of the impurities component, agriculture waste valorization of AE-RSC, AE-CCC, AE-PLC and AE-PPC samples reported that higher cellulose concentrations, ranging from 78.07 to 82.69% (w/w), were attained in alkaline-treated samples. The increase in the cellulose content was reported in agricultural waste samples subjected to alkaline-assisted extraction after the elimination of lignin and hemicellulose impurities [The extraction yield and chemical composition of different types of raw material and extracted cellulose samples are presented in residues ,29. The purities . The finThe appearance and microstructural morphology of AE-RSC, AE-CCC, AE-PLC, AE-PPC, and COM-C samples are displayed in −1 in all the raw materials, alkaline-extracted cellulose and commercial cellulose. The observed band characterized by O-H stretching vibrations could be correlated to the presence of aliphatic moieties in polysaccharides [−1 for commercial cellulose, while a similar vibration was noticed in raw material samples at 2920–2899 cm−1 and extracted cellulose samples at 2917–2896 cm−1. These band ranges are related to the general organic component of the polysaccharides [−1 and 1510 cm−1 corresponded to the C-O stretching vibrations of hemicellulose, and the C=C vibrations in lignin were not displayed in the observed spectra of extracted cellulose and commercial cellulose samples. The absence of absorption peaks at the aforementioned bands revealed that hemicellulose and lignin impurities were completely removed [−1 for all samples. The absorption peaks at wavenumber 1058 and 897 cm−1 are related to the C-O-C and C-O stretching at the β-glycosidic linkages [−1 in the cellulose samples showed increased cellulose contents after chemical treatment [FTIR analysis is a valuable method for studying the structural and physicochemical characteristics of polysaccharides. The FTIR spectra of different raw materials  and extracted cellulose samples using the alkaline process are given in charides . The C-Hcharides . The pea removed ,34. The linkages . In addireatment . The finThe XRD spectra of crude cellulose  and alkaline-extracted cellulose samples of different agricultural by-products are provided in max) of the AE-RSC, AE-CCC, AE-PLC, AE-PPC, and COM-C samples were obtained at 360, 364, 357, 358, and 357 °C, respectively , Tmax (364.67 °C) with residue at 600 °C (13.37%).Corncob, rice straw, Phulae pineapple leaves and peel waste valorization with prior bleaching and alkaline treatment yielded higher cellulose contents compared to the crude waste samples without any treatment. The AE-CCC sample showed lowest amounts of hemicellulose and lignin, followed by the highest extraction yield (38.18%) and α-cellulose (82.69%). Microstructure displayed the fibrous cellulose structure in AE-RSC and AE-PLC samples and a flaky structure was visualized in AE-CCC and AE-PPC samples that resembled the COM-C sample. The FTIR confirmed that, after bleaching and alkaline treatment, the main component in samples is cellulose. The XRD of all alkaline-treated samples presented an increased CI compared to the untreated raw material. The highest CI value was attained in AE-CCC, probably due to the sufficient elimination of hemicellulose and lignin, which are known as amorphous components. TGA and DTG results reported the excellent thermal stability of all the alkaline-processed cellulose samples in which AE-CCC showed the highest stability at TTherefore, this cellulose, extracted via alkaline extraction generated from the agricultural wastes, should be further investigated at the nano-scale level for the development of intelligent food packaging material, subject to the assessment of legal standards to claim it as an eco-friendly substitute to plastic packaging in the quality preservation of perishable food products. This may also eradicate the problem of burning agricultural wastes in crop fields, which causes a high rise in particulate matter of up to 2.5 due to the amount of smoke leading to environmental pollution and health concerns in the local community of Chiang Rai, Thailand. Moreover, further work should be conducted to demonstrate the waste utilization process as a vital asset to the economy of local farmers.